Molecular Ecology Notes (2004) 4, 680–682 doi: 10.1111/j.1471-8286.2004.00783.x © 2004 Blackwell Publishing Ltd Blackwell Publishing, Ltd. PRIMER NOTE Development of DNA microsatellite markers in the Andean root crop arracacha: Arracacia xanthorrhiza Banc. (Apiaceae) E. MORILLO,*† G. SECOND,† J. L. PHAM† and A. M. RISTERUCCI‡ * Departamento Nacional de Recursos Fitogenéticos y Biotecnología (DENAREF), Instituto Nacional Autónomo de Investigaciones Agropecuarias (INIAP), Quito, Ecuador, † IRD (Institut de Recherche pour le Développement), UMR 1097 DGPC/DYNADIV, BP 64501, 34394 Montpellier Cedex 5, France, ‡ CIRAD-AMIS/BIOTROP UMR 1096, 34398 Montpellier, France Abstract A microsatellite-enriched library was constructed in the Andean root crop arracacha ( Arracacia xanthorriza B). Of 18 loci tested, 14 were found to be polymorphic after screen- ing for diversity in different cultivars and related wild forms. Allelic diversity in the crop was low but the transferability of the primers to closely related wild forms was good. The loci reported here are the first genetic markers to be published for this species and will be useful for future germplasm characterization and studies of genetic diversity. Keywords: Andean crop, arracacha, Arracacia xanthorrhiza, microsatellites, zanahoria blanca Received 14 July 2004; revision accepted 3 August 2004 Arracacha (Arracacia xanthorrhiza B., tetraploid species: 2n = 4x = 44) is a vegetatively propagated carrot and the only Apiaceae species domesticated in the New World. Among nine minor Andean root and tuber species, arracacha is considered the most promising crop (Hermann 1997). To date, its genetic variability has been estimated using morphological characteristics, isozymes and random ampli- fied polymorphic DNA (RAPD) markers, leading to con- trasting conclusions. In order to study the genetic structure of the cultivated pool and its relation to wild relatives ( A. xanthorrhiza species complex; Randers et al . 2003), we have isolated and characterized microsatellite markers, which we present here. The microsatellite-enriched library was built following the procedure of Billote et al . (1999). Total genomic DNA was extracted from a white root cultivar using the PEX method (Jhingan 1992). Prior to amplification, DNA was digested with Rsa I and ligated with adapters (5 ′ -CTCTT- GCTTACGCGTGGACTA-3 ′ and 5 ′ -TAGTCCACGCG- TAAGCAAGAGCACA-3 ′ ). Selection of simple sequence repeat (SSR) fragments was carried out with a biotinylated SSR primer. Selected fragments were cloned into a pGEM- T plasmid (Promega A3600) and transformed in Epicurian- coli XL1-Blue MRF ′ supercompetent cells (Stratagene). In total, 192 white transformant clones were transferred to Hybon-N + nylon membranes (Amersham), and hybrid- ized using labelled P32 microsatellite oligoprobe (GA) 15 and (GT) 15 . For the 74 clones that gave a satisfactory posi- tive signal, the insert was sequenced in one direction. DNA sequence alignments and primer design were performed using dnastar 1.2. Primer pairs were synthesized for 26 microsatellite sequences of which 18 amplified the expected size fragment. SSR amplification was performed on 58 DNA samples of arracacha from Ecuador. SSR products were analysed following the protocol of Roy et al . (1996) applied with the automated infrared fluorescence technology of a sequencer (IR2; LI-COR Biosciences). For a given SSR locus, the forward primer was 5 ′ -end labelled with a M13 extension (5 ′ -CACGACGTTGTAAAACGAC-3 ′ ). Amplifi- cations were performed in 10 μ L of 20 ng genomic DNA, 0.02 μ m of the M13-labelled primer, 0.3 μ m of the reverse primer, 0.06 μ m of M13 primer-fluorescent dye IR700 or IR800, 2.5 m m MgCl 2 , 0.2 mm dNTP and 0.5 U Taq DNA polymerase (Promega). A Biometra thermocycler was used with the following cycling conditions: 94 ° C (1 min), 30 cycles at 94 ° C for 60 s, 45 – 60 ° C for 60 s, 72 ° C for 60 s and a final elongation step of 72 ° C for 7 min. IR700 or IR800- labelled polymerase chain reaction (PCR) products were diluted 10-fold in its loading buffer, subject to electro- phoresis in a 6.5% polyacrilamide gel and then sized by the Correspondence: E. Morillo, IRD (Institut de Recherche pour le Développement), UMR DGPC/DYNADIV, 911 Av. Agropolis BP 64501, 34394 Montpellier, Cedex 5, France. E-mail: morillo@mpl.ird.fr or denaref@ecnet.ec